JBRA Assist. Reprod. 2011;15(5):26-29
ARTIGO ORIGINAL
doi: 10.5935/1518-0557.2011.15.5.04
Effects of washed and autoclaved mineral oil overlay used during IVM of bovine oocytes on steroid concentration and preimplantational embryo development.
Department of Obstetrics and Gynecology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
1,4PhD, Post-doc of Department of Obstetrics and Gynecology, Faculty of Medicine of Ribeirão Preto, University of São Paulo
2PhD, Vitrogen Biotecnologia da Reprodução, Cravinhos - SP, Brazil
3Masters degree of Department of Obstetrics and Gynecology, Faculty of Medicine of Ribeirão Preto, University of São Paulo
5,6MD-PhD, Department of Obstetrics and Gynecology, Faculty of Medicine of Ribeirão Preto, University of São Paulo
ABSTRACT
Although droplet method has been widely used in IVF laboratories, the toxic contamination produced by deterioration of mineral oil quality and extraction of steroids from the culture medium into the oil overlay has been associated with impaired in vitro development and freezability of oocytes and embryos. The use of washed and autoclaved oil with 0.9% saline may provide a useful tool to prevent toxic contaminants in commercial mineral oil, but its effect upon steroids extraction from the culture medium remains to be determined. This study was designed to compare the concentration of steroids in IVM droplets of cumulusoocyte complexes (COCs) cultured under two types of oil overlay: washed and autoclaved mineral oil (WMO) or mineral oil in its commercial formulation (CMO) and to evaluate its influence on subsequent embryo development. Bovine COCs were cultured at a density of 1/10 µL in TCM-199 supplemented with estradiol-17β at 38.5 °C, 5% CO2 and 100% humidity during 24 h. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination (hpi). Media were collected before and after IVM, and stored at -20 °C. E2 and P4 were determined by chemiluminescence and embryo development was evaluated by cleavage, blastocyst and hatching rates at 168-224 hpi. E2 and P4 were dramatically reduced (P<0.01) in both oil overlay. Remaining levels of E2 in IVM microdrops were significantly higher in WMO (313.03 ± 72.6 ng/mL) than CMO (123.5 ± 46.1 ng/mL) showing lower steroid absorption by WMO overlay (P<0.01). P4 secreted by COCs was almost totally absorbed by mineral oil, irrespectively to the oil preparation. There were significantly lower hatching rates in WMO than CMO (71.4 % vs 94.4%, respectively; p<0.05). The use of hydrated mineral oil during IVM influences E2 bioavailability and may be detrimental to in vitro embryo development.
Keywords: Reproductive techniques, Steroids, IVM, Mineral oil, preimplantational embryo development
RESUMO
Embora o método de gotas seja amplamente utilizado em fertilização in vitro, a contaminação tóxica produzida por deterioração da qualidade do óleo mineral e extração de esteróides a partir do meio de cultura tem sido associada a insuficiente desenvolvimento in vitro e congelabilidade de oócitos e embriões. O uso do óleo lavado e autoclavado com solução salina 0,9% pode ser útil para evitar contaminantes tóxicos em óleo mineral comercial, mas seu efeito sobre a extração dos esteróides do meio de cultura continua indeterminado. Este estudo busca comparar a concentração de esteróides em gotículas de complexos cumulus-oócitos (COCs) em IVM cultivadas em dois tipos de sobreposição de óleo: lavado e autoclavado (WMO) ou óleo mineral em sua formulação comercial (CMO), além de avaliar sua influência sobre o desenvolvimento embrionário subseqüente. COCs bovinos foram cultivados a uma densidade de mL 10/01 em TCM-199 suplementado com E2 a 38,5 ° C, 5% de CO2 e umidade de 100% durante 24 h. Após a fecundação, os prováveis zigotos foram co-cultivados com células cumulus até 224 h pós-inseminação (hpi). Meios foram coletados antes e depois IVM, e armazenadas a -20 ° C. E2 e P4 foram determinados por quimiluminescência e desenvolvimento embrionário de blastocisto foi avaliada por clivagem e taxa de eclosão em 168-224 hpi. E2 e P4 estiveram drasticamente reduzidos (P <0,01) em ambas opções. Demais níveis de E2 em microgotas IVM foram significativamente maiores em WMO (313,03 ± 72,6 ng / mL) que no CMO (123,5 ± 46,1 ng / mL), mostrando menor absorção de esteróides na primeira (P <0,01). P4 secretado pelos COCs foi quase totalmente absorvido pelo óleo mineral, independentemente da preparação. Houve taxa de eclosão significativamente menor no WMO (71,4%) vs 94,4%, CMO, p <0,05). O uso de óleo mineral hidratado durante IVM influencia a biodisponibilidade do E2 e pode ser prejudicial para o desenvolvimento do embrião in vitro.
Palavras chaveTécnicas reprodutivas, esteroides, maturação in vitro, óleo mineral, desenvolvimento embrionário pré-implantação
INTRODUCTION
Culture system is a crucial step of assisted reproductive technologies influencing the potential of oocyte development and embryo implantation, which are detrimental for the success of in vitro fertilization (IVF) programs. Routinely, mammalian cumulus-oocyte complexes and embryos have been culture in microdroplet culture system. This method consists on a small volume of medium (<0.05mL) overlaid with mineral oil, which prevents evaporation of water from the medium and reduces gas and temperature fluctuation in the medium (Miller et al, 1994). Although widely used in IVF laboratories, the long-term culture of oocytes and embryos in small medium droplets have been received many concerns. The toxic contaminants released into the culture media during the deterioration of oil quality by exposure to light and heat, was associated with impaired in vitro development and freezability of oocytes and embryos (Erbach et al, 1995, Provo et al, 1998, Van Soom et al, 1994, Otsuki et al, 2007). Furthermore, it is known that exogenous steroids used as supplements or secreted into the culture medium and liposoluble substances in general may diffuse to the mineral oil, altering the hormonal concentrations in the microenvironment for the development of oocytes and embryos (Miller et al, 1987).
The negative effects of the use of mineral oil into the oocyte and embryo culture system may be prevented or minimized by the purification of the commercial mineral oil prior it use, which consists in equilibrate and autoclaving the oil with 0.9% physiological saline (. Borque et al, 1996, Van Soom et al, 2001). Performed in order to hydrate the oil and to transfer possible toxic traces of water-soluble compounds from the oil to the aqueous phase from which the oil is decanted, this procedure may propitiate an alternative tool to avoid further contamination of the culture system by the mineral oil in its commercial formulation.
Since it is recognized that oocytes require a specific steroid environment to achieve full maturation and developmental competence in vivo and that the mineral oil extracts exogenous steroids hormones used as supplements or secreted into the culture medium it is reasonable to hypothesize that oocytes and embryos cultured in small medium droplets under mineral oil may have their developmental course compromised by changes in the culture media hormonal concentrations. In fact, the lower progesterone concentration introduced by oil overlay has been suggested to be the determining factor for a delay in the timing of pig oocyte IVM (Shimada et al, 2001).
In this study, we designed a simple and comparative experiment evaluating different IVM procedures that include the use of a hydrated mineral oil overlay or mineral oil in its commercial formulation on steroid hormones concentration in the IVM medium. The preimplantational embryo development of in vitro matured oocytes and fertilized embryos was also compared.
METHODS
All chemicals used during the study were from Sigma Chemical Co. (St. Louis, MO, USA), unless otherwise stated.
2.1. Collection and in vitro maturation of bovine oocytes
Bovine ovaries were collected at a slaughterhouse and transported to the laboratory in 0.9% physiological saline supplemented with 0.05 g/L streptomycin at 35º C. Follicles measuring 3-8 mm in diameter were aspirated with 21 G needles adapted to 20 mL syringes. COCs retrieved from the follicular aspirate were washed with TALP-HEPES medium (Gordon, 1994) containing NaCl, KCl, NaHCO3, NaH2PO4, Na-lactate, CaCl2.2H2O, MgCl2.6H2O, HEPES, Pen-Strep, Na-pyruvate, and BSA, pH 7.4, and examined for morphology under a stereomicroscope. Only oocytes with a homogeneous cytoplasm and with more than three layers of granulosa cells were used (degrees I and II). The pool of immature COCs was randomly allocated to one of following culture conditions, according preparation of mineral oil to use in the IVM system: 1) washed and equilibrated mineral oil with 0.9 % physiological saline (WMO) and 2) mineral oil in the commercial formulation (CMO). COCs were cultured in 200 µL microdrops of TCM-199 medium (Invitrogen-Gibco BRL, Grand Island, NY, USA), supplemented with 10% fetal calf serum (Invitrogen-Gibco BRL) inactivated at 56 ºC for 30 minutes, 0.2 mM pyruvate,10 μg/mL gentamicin, 0.5 µg/mL FSH (Folltropin, Bioniche Animal Health Canada Inc., Belleville, ON, Canada), 5 µg/mL LH (Lutropin V, Bioniche Animal Health) and 1 µg/mL 17β-estradiol on 35 mm Petri dishes covered with 3 mL of M8410 mineral oil at 38.5º C, in atmosphere with 5% CO2, for a period of 24 h. Each experiment was carried out with five replicates and the COCs were cultured at a density of 1/10 µL culture medium. Since P4 is not commonly added into the bovine oocytes IVM medium, a control medium without an oil overlay for determination of total P4 secreted by COCs after 24 h was established at the same IVM protocol and compare with microdrops culture.
2.2. In vitro embryo production
Mature oocytes were inseminated with frozen-thawed, motile sperm separated by swim-up (Parrish et al, 1988) using Sperm TALP HEPES medium (Gordon, 1994) supplemented with 6 mg/mL BSA fraction V. Fertilization was performed in 100 mL drops of Fert TALP medium (Gordon, 1994), supplemented with 20 mg/mL heparin and 6 mg/mL fatty acid-free BSA fraction V under mineral oil. Sperm concentration during fertilization was approximately 106 spermatozoa/mL and the sperm were co-incubated with 20 COCs for 18 h in a humid 5% CO2 atmosphere at 38.5 °C. After fertilization, oocytes were partially denuded by pipetting in TALP HEPES medium (10) and 20 presumptive zygotes with up to two or three layers of cumulus cells were cultured in 50 mL drops of CR2aa medium (Rosenkrans et al, 1994), supplemented with 10% FCS (Invitrogen Life Technologies) and 1 mg/ mL BSA under mineral oil in a humid 5% CO2 atmosphere at 38.5 °C. The culture medium was replaced by fresh medium every 48 h. Cleavage rate was evaluated 72 h post-insemination (hpi), blastocyst rate was evaluated 168-192 hpi, and hatching rates at 224 hpi. Cleavage and blastocyst rates were calculated on the basis of number of presumptive zygotes, and hatching rates were calculated from the number of presumptive zygotes and total blastocysts.
2.3. Hormone analysis
Progesterone and 17β-estradiol concentrations were determined by chemiluminescence using a commercial kit from Diagnostics Products Corporation (DPC), Immulite System, Los Angeles, CA, USA. Samples were removed from the culture media at time 0 (newly prepared culture medium) and after 24 h of IVM and stored in a freezer at -20 ºC until hormone assays. The determinations were carried out in duplicate, without extraction and all samples for each steroid were analyzed in a single assay in order to avoid inter-assay variability. The intra-assays coefficients of variation were less than 10%.
2.4. Statistical analysis
The results of steroids are reported as means ± SD. Data were analyzed statistically by ANOVA followed by comparison of the means by the Tukey test. Cleavage and 8-16 cell embryos at 72 hpi, and blastocyst rate were assessed by Chi-square. Hatching rates were evaluated by Fisher’s exact test with the level of significance set at p<0.05.
RESULTS
The E2 concentrations in newly prepared culture medium before IVM and in remaining media after 24 h of COC culture are shown in Table 1. Mineral oil in its commercial presentation (CMO) absorbed more E2 than WMO (P<0.01). P4 was not detected in medium at time 0 h. However, P4 secreted by COCs after 24h of culture in a control IVM medium without oil overlay was 22.80 ± 4.26 ng/mL. Regardless of mineral oil preparation, P4 concentrations after IVM were very low, suggesting its almost total migration from the microdrop to the oil (p<0.01; Table 1). There were no differences (p<0.05) in total cleavage and blastocyst rates among WMO and CMO methods (Table 2). However, the hatching rates of in vitro fertilized embryos were significantly decreased in WMO (P = 0.03; Table 2).
Table 1. Steroid concentrations in culture media before and after 24 h of IVM of bovine oocytes under different mineral oil overlay
Table 2. Effects of mineral oil overlay on embryo development of oocytes matured in vitro
DISCUSSION
In this study, variations on steroid hormones concentration in bovine oocytes IVM medium was investigated using two different mineral oil overlay into the microdroplet IVM culture system: (a) the conventional mineral oil in its commercial presentation and (b) the hydrated and autoclaved mineral oil. It was also evaluated the preimplantational embryo development success from oocytes matured in both IVM procedures. Irrespectively of oil preparation, E2 concentration in IVM medium was markedly reduced by as much as 70% after 24 h of culture and the P4 secreted by COCs was almost fully practically extracted from culture medium into mineral oil, being reduced by 95% of its original level (23 ng/mL vs 0.68 and 0.87 ng/mL, in a control oil-free medium and CMO or WMO, respectively). In a previous study, Zheng et al (2003) demonstrated that E2 concentration is reduced by as much as 50% during the first 4 h of culture of primate COCs (Rhesus monkey) in 50 µL microdrops of TCM-199 medium covered with 3.5 ml mineral oil in its commercial formulation, while P4 concentration was reduced by as much as 93% during the first 2 h of IVM. According to Miller and Pursel (1987), radio labeled estradiol, progesterone and androstenedione were reduced by 51, 89 and 77%, respectively, in medium drops under oil after 24 h of incubation.
Comparing hydrated to conventional mineral oil overlay, our study showed that after 24 hours of culture the E2 diffusion was 14% higher in CMO than WMO resulting in a higher E2 concentration in microdrops covered with WMO (313 n/mL vs 123.5 ng/mL, for WMO and CMO respectively), while P4 diffusion was about 2% greater in CMO than in WMO. This differential steroid segregation into the oil overlay type suggests that during IVM period the oocytes might be exposure to different hormonal levels within the different type of oil overlay applied. However, the implications of this observation in the oocyte competence remain to be clarified.
Although, there were no significant differences of IVM oil overlay and steroid hormones concentrations interaction on cleavage and blastocyst rates, decreased hatching rates of blastocysts were obtained from oocytes matured under WMO. These data would suggest a potential dose-effect of E2 on distal endpoints in the oocyte development. Exogenous E2 added to oocyte IVM medium seems to negatively impact oocyte maturation, taking to account that dose used was 1µg/mL and that remaining E2 in WMO was 2.5 fold higher than CMO. A critical analysis of literature data shows that there are no conclusive data about the association between steroid concentration in the IVM medium and its effect on mammalian oocyte nuclear and/or cytoplasmic maturation as well as its subsequent development (Ali e Sirard, 2003; Beker et al, 2002; Lattanzi et al, 2003; Li et al, 2004; Moreira et al, 2002). Shimada et al (2002) reported that high steroid hormones secreted by pig COCs (1.2 ng/mL estradiol, 5.3 ng/mL testosterone and 64.2 ng/mL progesterone) accelerated GVBD and activated p34cdc2 kinase and MAP kinase in oocytes matured without oil overlay in comparison with those of oocytes cultured under mineral oil and improved rate of early embryonic development to the blastocyst stage after IVF. Steroid hormones such as P4, E2, estradiol, androgens, meiosis activating steroid and other lipid classes such as prostaglandins are secreted by cumulus cells when stimulated with gonadotropins during oocyte IVM (Armstrong et al, 1996; Gurevich et al, 1993) and all them can be absorbed by mineral oil or paraffin oil during the IVM. A recent study in human suggested aberrant DNA methylation at the imprinted H19 gene in oocytes following in vitro maturation (IVM) (Borghol et al, 2006) and that extraction of E2 from culture medium by mineral oil may also play a role in altered imprinting establishment (Anckaert et al, 2009).
Since the differential hormonal environments in which oocytes mature induce subtle alterations in meiosis kinetics in different mammalian species and appears to affect embryo development, this could have implications for the human ART-clinic, where different ovarian hyperstimulation protocols were shown to significantly influence endocrine parameters in serum and follicular fluid (Smitz et al, 2007) and where the upcoming technique of IVM of immature oocytes used mineral oil overlay (Mikkelsen et al, 2000; Segers et al, 2008).
CONCLUSION
In standard IVM microdroplet system, regardless of oil preparation, E2 and P4 concentrations were reduced by as much as 70 to 90% of the original hormonal levels. These results underscore the need to test culture conditions other than employ oil overlay or a two-step IVM method and to determine the exact steroid doses that should be used to induce human oocyte IVM.
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